Thrombin is a serine protease which serves as an active component in several hemostasis products. For example, fibrin sealants typically comprise a fibrinogen component and a thrombin component. When both components are mixed (e.g. when applied to a bleeding wound or surgical incision) thrombin cleaves fibrinogen and a fibrin polymer is formed. Concentrated purified thrombin in liquid form displays a reduction in activity during prolonged storage, mostly as a result of autolysis.
Hemostatic formulations containing liquid thrombin have special handling requirements in order to maintain thrombin's biologic activity and prevent autolytic degradation. For example, a liquid thrombin formulation requires refrigeration or the addition of protease inhibitors to maintain shelf-life stability. In the clinic, refrigeration is not always feasible, and promiscuous protease inhibitors may adversely affect the activity of thrombin.
Thrombin may be made into a lyophilized medical preparation, which is used after dissolving at the time of use. However, liquid preparations are advantageous as compared with the lyophilized preparations in that they can be easily administered without the additional step of dissolving in a solvent prior to use.
Other known compositions and methods for stabilizing thrombin are unsatisfactory and include the following: inclusion of various non-specific components (e.g. bulk carrier proteins such as albumins, different stabilizing sugars, general protease inhibitors etc.); formulation of the thrombin with inhibitors of thrombin activity, which although may be efficient, may also inactivate or inhibit the thrombin during use thereby reducing its effectiveness. In order to avoid or reduce inhibition, in use, it may be needed prior to use to dilute the inhibitor and therefore the thrombin. Formulation of a low dose thrombin, necessitates administration of larger amounts of the formulation.
International Patent Application Publication No. WO2008157304 discloses methods for stabilizing thrombin solutions with a preservative selected from benzyl alcohol or chlorobutanol and sucrose. Additional Patent Publications provide compositions comprising thrombin and non-specific inhibitors, and therefore, cannot effectively counter the thrombin-thrombin autolysis effect. For example, U.S. Pat. No. 4,409,334 discloses a stabilized thrombin preparation in solid or dissolved form comprising thrombin and as a stabilizer serum albumin together with at least one protease inhibitor which does not inhibit thrombin itself and at least one hexaligand chelate former.
European Patent No. EP0277096 B1 provides a stable thrombin composition containing purified thrombin, a polyol, and a buffer which contains either acetate or phosphate ions, wherein the preparation has a pH of about 5.0 to about 6.0.
European Patent No. EP 0478827 B1 provides a stable thrombin composition which includes a mixture of three stabilizers: HEPES-buffer, thiomersal, gelatin obtained by partial hydrolysis of collagen, and optionally Polybrene.
U.S. Pat. No. 7,351,561 discloses a stable thrombin preparation comprising thrombin and benzamidine or p-aminobenzamidine as stabilizer, and further including calcium chloride or sodium chloride as stabilizer, at least one buffer substance, and at least one of histidine, mannitol, sodium succinate, sodium lactate or arginine.
US Patent Application Publication No. 20090136474 (U.S. Pat. No. 8,394,372) provides a stabilized serine protease composition which comprises a serine protease; a reversible inhibitor of said serine protease (e.g. benzamidine, N,N-diethylethylenediamine, aminobenzamidine); and a stabilizing agent (e.g. 3-(N-morpholino)propane sulfonic acid). Non-patent literature describing various aspects of thrombin include: Pozzi N, et al., (2011) “Rigidification of the autolysis loop enhances Na(+) binding to thrombin” (Biophys Chem. 159(1):6-13); Marino, F. (2010) “Engineering thrombin for selective specificity toward protein C and PAR1” (J Biol Chem. 285(25):19145-52); Bah A, et al., (2009) “Stabilization of the E* form turns thrombin into an anticoagulant” (J Biol Chem. 24;284(30):20034-40); Yang L (2004) “Heparin-activated antithrombin interacts with the autolysis loop of target coagulation proteases” (Blood. 104(6):1753-9); and Rydel T J, et al (1994) “Crystallographic structure of human gamma-thrombin” (J Biol Chem. 269(35):22000-6).
Therefore, there remains a need for specific compounds useful to stabilize thrombin from autolytic degradation while retaining its biological activity. Preferably, the compounds may be used with a concentrated liquid thrombin formulation.